Anopheles mosquitoes are major malaria vectors, encompassing several species complexes with diverse life histories, transmission risks, and insecticide resistance profiles that challenge malaria control efforts. This study investigated the genetic structure and insecticide resistance profiles of Anopheles gambiae complex mosquitoes in Tanzania. We analysed whole-genome sequence data of 300 mosquitoes collected between 2012-2015 across four regions in northern Tanzania and identified An. gambiae s.s., An. arabiensis, and a distinct taxonomic group that was previously unknown. This distinct taxon has a unique profile of genetic diversity, and appears restricted to the coastal region, and we refer to it as the Pwani molecular form. Analysis of insecticide resistance based on target-site mutations and copy number variations (CNV) showed that these markers were strikingly absent from the Pwani molecular form in contrast to other taxa. Our analysis also revealed a pattern of geographical isolation in the An. gambiae s.s. populations, with samples from north-western site (Muleba) clustering separately from those collected in the north-eastern site (Muheza). These geographically isolated sub-populations also had differing resistance and selection profiles, with An. gambiae s.s. from the north-western site showing genomic evidence of higher resistance to pyrethroids compared to the north-eastern population. Conversely, An. arabiensis showed no geographical population structuring, with a similar insecticide resistance profile across all sampling locations, suggesting unrestricted gene flow. Our findings underscore the need to incorporate genetic data into malaria vector surveillance and control decisions; and could inform the development and deployment of new interventions.

Insecticide resistance provides both a pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses. Malaria control remains heavily dependent on deployment of insecticides, primarily in long lasting insecticide treated nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years. Identifying genetic mechanisms causing high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes data we identified a very recent selective sweep in Ugandan mosquitoes which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a point mutation in Cyp6p4, an insertion of a partial Zanzibar transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region, with stepwise replacement of the double-mutant (Zanzibar TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids; is strongly predictive of resistance to pyrethroids but importantly, appears less effective against LLINs co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable even within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries.